DESCRIPTION: The long-term objective of this program is to understand the mechanism and molecular pharmacology of the neuroendocrine-type KATP channels involved in the pathogenesis of Neonatal and Type 2 Diabetes. Normal ABCC8/KCNJ11-encoded, MgADP-stimulated/ATP-inhibited (SUR1/KIR6.2)4 KATP channels regulate insulin secretion and glucose homeostasis. Since loss-of-activity mutations in ABCC8 were found in infants with persistent hyperinsulinemic hypoglycaemia we hypothesized that gain-of-activity mutations in ABCC8 cause Neonatal Diabetes (ND). In collaboration with an international network for the study of ND for genetic diagnosis we discovered multiple novel heterozygous mutations in ABCC8 in ND patients. The genetic evidence for the causal role of these mutations is strong. They map to SUR1 domains that may control channel activity. Our preliminary analyses indicated NDSUR1/KIR6.2 channels are hyperactive in living cells. Our first aim is to complete the verification that mutant receptors, NDSUR1, significantly increase the open channel probability (PO) in intact mammalian cells. NDSUR1 hyperactivated the channel in physiologic Mg-nucleotides in isolated membrane fragments. We propose to define the principal mechanisms of hyperactivation. We have shown previously that beyond stimulating the channel in the presence of MgATP/ADP, SUR1 controls the maximal PO in the absence of nucleotides, and increases the apparent affinity of KIR6.2 for inhibitory ATP. Therefore, in the second aim, we will test our hypothesis that there are three mechanisms of hyperactivation: an increase in the Mg-nucleotide-dependent stimulatory action of SUR1, an increase in the nucleotide-independent POmax, and a decrease in the sensitivity of the channel to inhibitory ATP with no change in ligand-independent gating. In heterozygous NDABCC8 patients KATP pores interact with both NDSUR1 and SUR1 subunits. Thus our third aim is to establish the dependence of hyperactivation on the number of NDSUR1 subunits in heterozygous channels. Tolbutamide (~10-4 M) reduced the activity of NDSUR1 channels, indicating that many NDABCC8 cases are treatable with oral sulfonylureas instead of daily insulin injections. Thus our fourth aim is to determine whether any of the hyperactivating NDABCC8 mutations compromise high-affinity sulfonylurea binding or its coupling to the KATP pore-closing machinery. To achieve these four interconnected specific goals we employ the patch-clamp method, single-channel kinetics analysis, concatenated subunits, structural modeling, and affinity photolabeling techniques. The outcomes of this study will deepen our understanding of the novel mechanism of metabolism-excitation uncoupling and improve the lives of ND patients, which is highly relevant to the mission of NIGMS and NIDDK. PUBLIC HEALTH RELEVANCE: As the pandemic of diabetes continues one in three children born in 2000 will be diagnosed with diabetes in their lifetime. The difficulty of treating this disorder stems in part from our insufficient understanding of the molecular mechanisms that play key roles in regulating insulin secretion. Recently we discovered that mutations in the key regulator of insulin release, sulfonylurea receptor 1, cause diabetes within the first months of life. The proposed studies of these mutant receptors will reveal a novel diabetogenic mechanism and result in a new and better treatment of neonatal diabetes.